In pathology, deparaffinization and antigen retrieval pretreatment for immunohistochemical staining of a glass slide with a tissue specimen usually includes, as described in Non-patent Publication 1: the first step of deparaffinization by passing a formalin-fixed paraffin-embedded tissue section through an organic solvent layer, such as xylene, benzene, or toluene, three times for three-minutes each; the second step of rehydration of the glass slide and the tissue specimen by passing them through an amphiphilic solution, such as ethanol, four times for three-minutes each; and the third step of antigen retrieval of the rehydrated tissue specimen by heat treating the rehydrated tissue specimen immersed in an antigen retrieval solution, such as a citrate buffer or a Tris-EDTA solution.
The organic solvents used in the first step of the pretreatment, such as xylene, benzene, or toluene, are highly toxic and volatile, which requires exhaust equipment such as draft, and disadvantageously affects the environment of engaged technicians.
As a substitute for the disadvantageous organic solvents, highly safe deparaffinization agents are commercially available, such as Hemo-De (trade name, manufactured by FALMA), an organic solvent extracted and purified from citrus peels, Clear-Plus and Hemo-Clear (both trade names, manufactured by FALMA) using aliphatic hydrocarbon (alkanes), and Tissue Clear (trade name, manufactured by SAKURA FINETEK JAPAN CO., LTD.).
Rather than using the highly safe deparaffinization agents, more convenient methods are desired wherein the three steps of the pretreatment are performed simultaneously in a single solution, and some proposals have been made.
For example, Patent Publication 1 proposes a pretreatment process wherein deparaffinization and antigen retrieval are performed simultaneously by heating, to a temperature higher than the melting point of paraffin, a paraffin-embedded tissue specimen in a solution containing a paraffin-solubilizing organic solvent selected from aromatic hydrocarbon, a terpene, or isoparaffinic hydrocarbon, a polar organic solvent, an antigen retrieving component, and a surfactant. Patent Publication 1 also discloses that, for removing residual surfactant in a washing step following the pretreatment process, cyclodextrin, which binds to the surfactant, may be contained in the washing solution.
On the other hand, Patent Publication 1 discloses in Example 7 that, when the above-mentioned solution used in the pretreatment is used consecutively, the residual paraffin eluted in the solution after the pretreatment adheres to a next slide, so that this solution is not suitable for consecutive use.
Further, when a solution of the composition disclosed in Patent Publication 1 is made to have an alkaline pH, in the immunohistochemical staining following the pretreatment, the glass slide with the tissue specimen adhered thereto is prone to repel the staining reagent at portions free of the tissue specimen, resulting in uneven staining.
Patent Publications 2 and 3 disclose that deparaffinization and antigen retrieval may be effected simultaneously using a solution containing various buffers, surfactants, ethylene glycol, and the like, in the same way as the pretreatment process disclosed in Patent Publication 1.
However, in these publications, consecutive use of the pretreatment solution is not intended either, similarly to the solution of Patent Publication 1, and there is no description of a technology such as for dispersing the eluted paraffin in the solution, or the like. Thus it is likely that the pretreatment solutions disclosed in these publications, when used consecutively, also have the same problems as that of Patent Publication 1.
Incidentally, some pretreatment solutions for immunohistochemical staining which effect deparaffinization and antigen retrieval simultaneously are already in the market, such as those disclosed in the above patent publications, including Trilogy (trade name, manufactured by Cell Marque Corporation) for single use, or Target Retrieval Solution pH9 (3-in-1) (trade name, manufactured by DAKO) for use three consecutive times within a week.
The compositions of these commercial products are not known, but do not contain cyclodextrin. Further, in all of such products, hydrophobic paraffin eluted into the pretreatment solution during use often floats on the solution surface.
Such insoluble paraffin on the solution surface adheres to a glass slide with a tissue specimen when it is taken out of the solution, so that more washing steps are required before the subsequent staining, which complicates the operation. Otherwise, the insoluble paraffin adversely affects the subsequent immunohistochemical staining. Further, when staining is performed in an automatic immunohistochemical stainer, the insoluble paraffin may adversely affect the stainer.